J Life Sci Res 2019; 17: 7-10
© 2019 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Short Communication

Influence of the spacer arm length of biotinylation reagent for preparing abiotinylated microplate for biotin assay
Makoto MURATSUGU † and Kouichi NISHIDA
Bioanalytical Science Laboratory, Department of Clinical Nutrition, Osaka Prefecture University, 3-7-30 Habikino, Habikino, Osaka583-8555, Japan.
Received 4 March 2019; accepted 29 August 2019
We directly introduced biotin moieties into an amino group-rich microplate using biotinylation reagents. Thebiotinylated microplate could be successfully used for a solid phase-supported ligand competitive assay for biotin. The
distance between the biotin-immobilized solid phase and the ureido ring of biotin was called the bridge length in thisstudy. Examining biotinylation reagents with various spacer arm lengths led to a good dose-response curve as the bridge
length increased. This result, supported by computer simulation, suggested that the steric hindrance of enzyme-labeledstreptavidin affected its binding to immobilized biotin.
Key words: biotinylation reagent; biotinylated microplate; competitive assay; computer simulation

been used as biotinylated proteins.10

1 Introduction
Biotin is a water-soluble vitamin and a growth factor for

In this study, we directly bound biotin to a microplate

microorganisms and animals, 1 and is widely distributed in

and investigated the influence of the spacer arm length offoods.2 A variety of methods for measuring biotin have been

biotinylation reagent for preparing a biotinylated microplate.

developed, and the biotin contents of natural materials havebeen determined. Many microorganisms that require biotin

2 Materials and Methods

as a growth factor have been used for detecting biotin, and a

2.1 Materials

turbidimetric method using Lactobacillus plantarum has been

The following materials were obtained from the sources

widely used.3-5 An agar plate method using L. plantarum wasindicated: flat-bottomed multiwell microplates (96 wells,also developed, offering greater precision than the turbidimetric

Summilon A, Aminoplate) and ELISA Color Reagent kit

method.6(Type O) from Sumitomo Bakelite Co., Ltd., Tokyo, Japan;

Using the high affinity of avidin or streptavidin for

N-hydroxysuccinimidobiotin (NHS-biotin), sulfosuccinimidyl-

the ureido ring of biotin, a solid phase-supported receptor

6-biotinamido-hexanoate (Sulfo-NHS-LC-biotin), succinimidyl-

competitive assay and a solid phase-supported ligand

6'-(biotinamido)-6-hexanamido hexanoate (NHS-LC-LC-biotin),

7Inand biotin pentafluorophenyl-ester (PFP-biotin) from Pierce

the former assay, biotin and enzyme-labeled biotin are added

Chemical Co., Rockford, IL, U.S.A. (Fig. 1); biotin, horseradish

almost simultaneously to the avidin-immobilized solid phase

peroxidase (HRP)-conjugated streptavidin (HRP-streptavidin),on a microplate, and unbound biotin and labeled biotin arebovine serum albumin (BSA), and dimethyl sulfoxide (DMSO)

removed. In the latter assay, free biotin is first added to thefrom Sigma Chemical Co., St. Louis, MO, U.S.A. All otherbiotin-immobilized solid phase, the enzyme-labeled avidin or

chemicals were of reagent grade or better. The water used wasstreptavidin is immediately added, and the biotin-bound enzyme-

17-Mohm grade.

labeled avidin or streptavidin is finally removed. To immobilize

2.2 Biotinylated microplatecompetitive assay have been developed as biotin assays.

biotin on the solid phase, biotinylated proteins are generally

NHS-biotin, Sulfo-NHS-LC-biotin, NHS-LC-LC-adsorbed onto a microplate. For example, biotinylated bovine

biotin, and PFP-biotin were used to biotinylate Aminoplate,serum albumin7-9 or goat anti-rabbit immunoglobulin G have

which is rich in amino groups on the surface of the plate. The
biotinylation reagents were dissolved in DMSO, then diluted†Corresponding author, Email: mmakoto@rehab.osakafu-u.ac.jp

with 0.1 mol L-1 sodium phosphate buffer, pH 7.4, containing

8

biotinylated microplate followed by 50 µL of 0.1 µg mL-1 HRP-O
HNF

[1]FO

FFOHNOOOO[3]

N[4]Fig. OO

Absorbance at 490 nm was then measured using the microplate
reader and the data were transferred to a computer using aOHNHNOOaddition of a reaction stop solution in the kit to each well.

SOO

20 minutes at 25°C, the color reaction was terminated by theNHOOFig. 1kit was added to each well. After the plate was incubated forON

microliters of substrate solution in the ELISA Color Reagent

OO
Nat 37°C, then washed three times with PBS-M. One hundred

SF

[2]

streptavidin in PBS-MB. The plates were incubated for 1 hour

NHNH

custom-made program for data analysis.11

NHS

3 Results and Discussion

OHN
HNO

NHS

Various long- and short-chain biotinylation reagents havebeen synthesized12 and can now be commercially purchased.
Aminoplates were biotinylated using biotinylation reagentswith varying spacer arm lengths. The lengths of PFP-biotin,

Structure of biotinylation reagents.

NHS-biotin, Sulfo-NHS-LC-biotin, and NHS-LC-LC-biotin[1] PFP-biotin (spacer arm length, 0.96 nm), [2] NHS-biotin(1.35 nm), [3] Sulfo-NHS-LC-biotin (2.24 nm), [4] NHSLC-LC-biotin (3.05 nm).

are 0.96, 1.35, 2.24, and 3.05 nm, respectively. After these-1

reagents reacted with amino groups on the solid phase, thedistance between the solid phase and the ureido ring of biotin

0.15 mol L NaCl. Sulfo-NHS-LC-biotin is soluble in aqueous

was designated as the bridge length in this study. When thesolution, but was dissolved in DMSO for comparison. One

bridge lengths for NHS-LC-LC-biotin, Sulfo-NHS-LC-biotin,

hundred microliters of the biotinylation reagent solution (1.5NHS-biotin, and PFP-biotin are represented by BL1, BL2, BL3,-5-1

× 10 mol L ) was added to each well of Aminoplate andand BL4, respectively, we have BL1 > BL2 > BL3 = BL4. The

incubated for 2 hours at 37°C. After the wells were washeddose-response curves were obtained using these biotinylated

three times with Dulbecco’s phosphate-buffered saline without

microplates (Fig. 2). A better dose-response curve was obtained

Ca2+ or Mg2+ (PBS) containing 0.02% NaN3 (PBS-N), 300 µL

as the bridge length increased. In addition, the binding of HRP-of 1% BSA in PBS-N was added to each well. The plates werestreptavidin increased as the bridge length increased (Fig. 3).

incubated for 2 hours at 37°C and washed three times with

These results are explained below. A good dose-response curve

PBS containing 0.05% merthiolate (PBS-M). The biotinylated

is obtained with an increase in immobilized biotin in the solid

Aminoplate was designated as a biotinylated microplate in thisphase-supported ligand competitive assay,7 but this may notstudy.

have been the case here. Since the amount of each biotinylation

2.3 Dose-response curves for a solid phase-supported

0.8

competitive assay

mL-1 HRP-streptavidin in PBS-M containing 0.1% BSA (PBSMB). The plates were incubated for 1 hour at 37°C, then washedthree times with PBS-M. One hundred microliters of substratesolution in the ELISA Color Reagent kit was added to each

Absorbance

Fifty microliters of dilutions of biotin was added to eachwell of a biotinylated microplate followed by 50 µL of 0.1 µg0.4well. After the plate was incubated for 20 minutes at 25°C, the0color reaction was terminated by the addition of a reaction stop
solution in the kit to each well. Absorbance at 490 nm was thenmeasured using a microplate reader (Model 450, Bio-Rad Labs.,

2.4 Binding of HRP-streptavidin on the biotinylated microplate

Fig. 2

An increase in HRP-streptavidin binding indicates anincrease of biotin uptake into a microplate, such that the HRPstreptavidin binding method can be used to evaluate biotin
uptake. Fifty microliters of PBS-M was added to each well of a102103[Biotin] × 1011/mol L-1

Richmond, U.S.A.) and the data were transferred to a computer
using a custom-made program for data analysis.11101

Fig. Effect of the bridge length on dose-response curves.

The bridge length is defined in the text. □, 1.5 × 10-5 mol
L-1 PFP-biotin; ○, 1.5 × 10-5 mol L-1 NHS-biotin; ■, 1.5× 10 -5 mol L -1 Sulfo-NHS-LC-biotin; ●, 1.5 × 10 -5 mol
L -1 NHS-LC-LC-biotin. 0.1 µg mL -1 HRP-streptavidin.
Vertical bars indicate the standard deviation from the meanof duplicate experiments.

Relative absorbance

92.0

B

B

HRP
StAv

HRP
StAv
B

HRP
StAv

B

B

B

1.00.5

B

B

HRP
StAv
B

HRP
StAv
B

B

B

HRP
StAv

0Fig. Fig. 3PFP-biotin

NHS-biotin

Sulfo-NHS-LC-biotin

NHS-LC-LC-biotin
B Biotin (ureid ring)

Effect of the bridge length on HRP-streptavidin binding
experiments.
-5StAv

Streptavidin

HRPHorse radish peroxidase
BridgeSolid phase-1

1.5 × 10 mol L PFP-biotin, NHS-biotin, Sulfo-NHSLC-biotin, and NHS-LC-LC-biotin. 0.1 µg mL -1 HRPstreptavidin. Relative absorbance means that the absorbancefor NHS-LC-LC-biotin is the standard value for comparison.
4Vertical bars indicate the standard deviation from Fig.
the meanof duplicate experiments.
-51.0-1

reagent used in this experiment was constant (1.5 × 10 mol L ),0.8

LC-LC-biotin are 410, 341, 557, and 568 Da, respectively), the[BsA*]

and these reagents are small molecules (the molecular masses
of PFP-biotin, NHS-biotin, Sulfo-NHS-LC-biotin, and NHS-Influence of the bridge length on the reaction of
immobilized biotin and HRP-streptavidin.

Fig. 0.4

in the solid phase could possibly be the same; therefore, the0.20.0

irrespective of the biotinylation reagent used. In contrast, biotin
is much smaller than HRP-streptavidin, the molecular massesof biotin, HRP, and streptavidin being 0.224, 440, and 680 kDa,
respectively. The large HRP-streptavidin demonstrated stericD= 0.7

0.6

collision frequency between these reagents and amino groups
number of immobilized biotin molecules appears to be the sameD= 1

Fig. 

hindrance of biotin binding (Fig. 4). When the bridge lengthD= 0.1

10-1

100 101
bf102

103

Dose-response curves for the solid-phase-supportedbiotin competitive assay: effect of the α coefficient.
K = 1015, a* = 1, bs = 10

is short, the number of HRP-streptavidin complexes bound toimmobilized biotin may decrease (Fig. 4).

Fig. 5small. The computer simulation is performed using Eq. 2 in

The idea that the steric hindrance of HRP-streptavidin

ExcelTM (Microsoft Co., Redmond, U.S.A.). Simulated plots

affected its binding to immobilized biotin was confirmedof [BsA*] as a function of bf when changing the values of α areby computer simulation using a mathematical model of theshown in Fig. 5. As α increases (decrease in steric hindrance ofassay (Appendix). The steric hindrance of HRP-streptavidin

HRP-streptavidin), the response curves improve. The computer

is considered to correspond to a decrease in the biotin groups

simulation supported the steric hindrance of HRP-streptavidin

immobilized on the solid phase. We introduced an α coefficientaffecting its binding to immobilized biotin. In order to confirm(0 < α ≤ 1) into Eq. A6 in the Appendix, that is:

this idea, it is necessary to experimentally compare the binding(1)

abs = [Bs] + [Bs A*].

Therefore, Eq. A8 is as follows,( 2)[Bs A*] =abs [ [K (bƒ + abs) + Ka* + 1] ‒amount of a radioisotope labeled-HRP-streptavidin 13,14 on abiotin-bound microplate with a long bridge to its amount on themicroplate with a short one. This is an issue for future research.

K2 (bƒ + abs)2 + 2K (1 ‒ 2Ka*) (bƒ + abs) + (Ka* + 1)2 ]2K (bƒ + abs),

where bf represents the total concentration of biotin in standards

Conflict of Interestor samples, and the intensity of color after an enzyme reaction

All the authors declare that they have no conflicts of interest.

is proportional to [BsA*]. As mentioned above, bs is considered
to be constant. If α is less than 1, it means that bs is apparently

10References
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Appendix
Biotin binds strongly to streptavidin, which has four binding sites. We assumed that each biotin molecule (ligand) bound to a
streptavidin molecule (receptor) in order to simplify the model. The reaction of the solid phase-supported ligand competitive assay forbiotin7 can be schematized in the following reaction schemes:(A1)

Bƒ + A*

Bƒ A*

(A2)

Bs + A*

Bs A*

where Bƒ, Bs, and A* represent free biotin, solid phase-supported biotin, and enzyme-labeled streptavidin, respectively. The equilibrium
constants for these reactions, schemes A1 and A2, are designated as Kƒ and Ks, respectively:

(A3)

Kƒ =

[Bƒ A*][Bƒ] [A*]

(A4)

Ks =

[Bs A*][Bs] [A*]The total concentrations of free biotin (bƒ), solid phase-supported biotin (bs), and enzyme-labeled streptavidin (a*) are given by(A5)bƒ = [Bƒ] + [Bƒ A*](A6)

bs = [Bs] + [Bs A*](A7)

a* = [A*] + [Bƒ A*] + [Bs A*]

Assuming Ks = Kf ( ≡ K), the following equation is derived from Eq. A3 to Eq. A7:(A8)[Bs A*] =bs [ [K (bƒ + bs) + Ka* + 1] ±K2 (bƒ + bs)2 + 2K (1 ‒ 2Ka*) (bƒ + bs) + (Ka* + 1)2 ]2K (bƒ + abs).